WO2023274403A1 - Use of a lrp1 inhibitor in treating notch signaling-dependent disease - Google Patents
Use of a lrp1 inhibitor in treating notch signaling-dependent disease Download PDFInfo
- Publication number
- WO2023274403A1 WO2023274403A1 PCT/CN2022/103347 CN2022103347W WO2023274403A1 WO 2023274403 A1 WO2023274403 A1 WO 2023274403A1 CN 2022103347 W CN2022103347 W CN 2022103347W WO 2023274403 A1 WO2023274403 A1 WO 2023274403A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lrp1
- lrpap1
- seq
- leukemia
- amino acid
- Prior art date
Links
- 108010070047 Notch Receptors Proteins 0.000 title claims abstract description 84
- 102000005650 Notch Receptors Human genes 0.000 title claims abstract description 83
- 230000011664 signaling Effects 0.000 title claims abstract description 51
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 35
- 201000010099 disease Diseases 0.000 title claims abstract description 34
- 230000001419 dependent effect Effects 0.000 title claims abstract description 29
- 239000003112 inhibitor Substances 0.000 title claims abstract description 21
- 101100075486 Caenorhabditis elegans lrp-1 gene Proteins 0.000 title description 13
- 208000032839 leukemia Diseases 0.000 claims abstract description 59
- 238000000034 method Methods 0.000 claims abstract description 32
- 239000003814 drug Substances 0.000 claims abstract description 14
- 229940079593 drug Drugs 0.000 claims abstract description 4
- 238000012216 screening Methods 0.000 claims abstract description 4
- 101000896414 Homo sapiens Nuclear nucleic acid-binding protein C1D Proteins 0.000 claims abstract 14
- 101001043564 Homo sapiens Prolow-density lipoprotein receptor-related protein 1 Proteins 0.000 claims abstract 14
- 102100021713 Nuclear nucleic acid-binding protein C1D Human genes 0.000 claims abstract 14
- 210000004027 cell Anatomy 0.000 claims description 146
- 150000001413 amino acids Chemical class 0.000 claims description 41
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 41
- 102100027165 Alpha-2-macroglobulin receptor-associated protein Human genes 0.000 claims description 39
- 101000836956 Homo sapiens Alpha-2-macroglobulin receptor-associated protein Proteins 0.000 claims description 38
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 38
- 229920001184 polypeptide Polymers 0.000 claims description 34
- 230000000694 effects Effects 0.000 claims description 25
- 230000014509 gene expression Effects 0.000 claims description 25
- 238000009739 binding Methods 0.000 claims description 19
- 239000003446 ligand Substances 0.000 claims description 19
- 230000027455 binding Effects 0.000 claims description 18
- 241000282414 Homo sapiens Species 0.000 claims description 17
- 208000029052 T-cell acute lymphoblastic leukemia Diseases 0.000 claims description 15
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 13
- 238000012217 deletion Methods 0.000 claims description 13
- 230000037430 deletion Effects 0.000 claims description 13
- -1 small molecule compound Chemical class 0.000 claims description 12
- 239000005557 antagonist Substances 0.000 claims description 11
- 238000006467 substitution reaction Methods 0.000 claims description 11
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 claims description 8
- 206010006187 Breast cancer Diseases 0.000 claims description 7
- 208000026310 Breast neoplasm Diseases 0.000 claims description 7
- 239000004055 small Interfering RNA Substances 0.000 claims description 7
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 claims description 6
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 6
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 claims description 6
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 5
- 201000011176 T-cell adult acute lymphocytic leukemia Diseases 0.000 claims description 5
- 201000005202 lung cancer Diseases 0.000 claims description 5
- 208000020816 lung neoplasm Diseases 0.000 claims description 5
- 108020005004 Guide RNA Proteins 0.000 claims description 4
- 206010025323 Lymphomas Diseases 0.000 claims description 4
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 4
- 108020004459 Small interfering RNA Proteins 0.000 claims description 4
- 201000007270 liver cancer Diseases 0.000 claims description 4
- 208000014018 liver neoplasm Diseases 0.000 claims description 4
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 4
- 102100038895 Myc proto-oncogene protein Human genes 0.000 claims description 3
- 239000002679 microRNA Substances 0.000 claims description 3
- 101710135898 Myc proto-oncogene protein Proteins 0.000 claims description 2
- 101100491597 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) arg-6 gene Proteins 0.000 claims description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 claims description 2
- 108091070501 miRNA Proteins 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 10
- 208000037819 metastatic cancer Diseases 0.000 claims 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 description 65
- 229940127276 delta-like ligand 3 Drugs 0.000 description 43
- 102000004169 proteins and genes Human genes 0.000 description 43
- 235000018102 proteins Nutrition 0.000 description 41
- 102100036466 Delta-like protein 3 Human genes 0.000 description 39
- 101000928513 Homo sapiens Delta-like protein 3 Proteins 0.000 description 39
- 206010028980 Neoplasm Diseases 0.000 description 25
- 108091030071 RNAI Proteins 0.000 description 23
- 230000009368 gene silencing by RNA Effects 0.000 description 23
- 230000037361 pathway Effects 0.000 description 20
- 208000005623 Carcinogenesis Diseases 0.000 description 16
- 102100023181 Neurogenic locus notch homolog protein 1 Human genes 0.000 description 16
- 108010029755 Notch1 Receptor Proteins 0.000 description 16
- 230000036952 cancer formation Effects 0.000 description 16
- 231100000504 carcinogenesis Toxicity 0.000 description 16
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 16
- 239000002953 phosphate buffered saline Substances 0.000 description 16
- 238000001262 western blot Methods 0.000 description 15
- 235000001014 amino acid Nutrition 0.000 description 14
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 14
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 13
- 230000006870 function Effects 0.000 description 13
- 239000000872 buffer Substances 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 12
- 210000004379 membrane Anatomy 0.000 description 12
- 239000012528 membrane Substances 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 11
- 238000010186 staining Methods 0.000 description 11
- 238000011529 RT qPCR Methods 0.000 description 10
- 230000003247 decreasing effect Effects 0.000 description 10
- 230000003993 interaction Effects 0.000 description 10
- 239000006166 lysate Substances 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- 238000013508 migration Methods 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 238000012986 modification Methods 0.000 description 10
- 102000007469 Actins Human genes 0.000 description 9
- 108010085238 Actins Proteins 0.000 description 9
- 101100074828 Caenorhabditis elegans lin-12 gene Proteins 0.000 description 9
- 239000011324 bead Substances 0.000 description 9
- 230000005012 migration Effects 0.000 description 9
- 239000005089 Luciferase Substances 0.000 description 8
- 230000007547 defect Effects 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 230000009545 invasion Effects 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 108060001084 Luciferase Proteins 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 210000004899 c-terminal region Anatomy 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- 238000010166 immunofluorescence Methods 0.000 description 7
- 150000002632 lipids Chemical class 0.000 description 7
- 230000004807 localization Effects 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 230000002018 overexpression Effects 0.000 description 7
- 230000004083 survival effect Effects 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 108010077544 Chromatin Proteins 0.000 description 6
- 238000000692 Student's t-test Methods 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 230000004709 cell invasion Effects 0.000 description 6
- 235000012000 cholesterol Nutrition 0.000 description 6
- 210000003483 chromatin Anatomy 0.000 description 6
- 238000010293 colony formation assay Methods 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 238000000386 microscopy Methods 0.000 description 6
- 230000004614 tumor growth Effects 0.000 description 6
- 208000002330 Congenital Heart Defects Diseases 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 101000843449 Homo sapiens Transcription factor HES-5 Proteins 0.000 description 5
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 5
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 5
- 241000124008 Mammalia Species 0.000 description 5
- 238000011789 NOD SCID mouse Methods 0.000 description 5
- 102100030853 Transcription factor HES-5 Human genes 0.000 description 5
- 230000006907 apoptotic process Effects 0.000 description 5
- 208000028831 congenital heart disease Diseases 0.000 description 5
- 230000003828 downregulation Effects 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 101150084157 lrp-1 gene Proteins 0.000 description 5
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000002894 organic compounds Chemical class 0.000 description 5
- 238000010379 pull-down assay Methods 0.000 description 5
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 101001002170 Homo sapiens Glutamine amidotransferase-like class 1 domain-containing protein 3, mitochondrial Proteins 0.000 description 4
- 101000843556 Homo sapiens Transcription factor HES-1 Proteins 0.000 description 4
- 229940122178 LRP1 antagonist Drugs 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 102100030798 Transcription factor HES-1 Human genes 0.000 description 4
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 230000005757 colony formation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 230000002121 endocytic effect Effects 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 201000003444 follicular lymphoma Diseases 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 201000005249 lung adenocarcinoma Diseases 0.000 description 4
- 108010082117 matrigel Proteins 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- DLWLXTLRGQWGPC-UHFFFAOYSA-N 10,13-dimethyl-17-[1-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]propan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-ol Chemical compound C1C=C2CC(O)CCC2(C)C(CCC23C)C1C3CCC2C(C)CNC1=CC=C([N+]([O-])=O)C2=NON=C12 DLWLXTLRGQWGPC-UHFFFAOYSA-N 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 3
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 3
- 108090000672 Annexin A5 Proteins 0.000 description 3
- 102000004121 Annexin A5 Human genes 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 108010040476 FITC-annexin A5 Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 208000017604 Hodgkin disease Diseases 0.000 description 3
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 3
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 3
- 101001013150 Homo sapiens Interstitial collagenase Proteins 0.000 description 3
- 208000031671 Large B-Cell Diffuse Lymphoma Diseases 0.000 description 3
- 108010061306 Lipoprotein Receptors Proteins 0.000 description 3
- 102000011965 Lipoprotein Receptors Human genes 0.000 description 3
- 208000025205 Mantle-Cell Lymphoma Diseases 0.000 description 3
- 102000000380 Matrix Metalloproteinase 1 Human genes 0.000 description 3
- 108010085220 Multiprotein Complexes Proteins 0.000 description 3
- 102000007474 Multiprotein Complexes Human genes 0.000 description 3
- 241000244206 Nematoda Species 0.000 description 3
- 108700037638 Neurogenic locus notch homolog protein 1 Proteins 0.000 description 3
- 102000001759 Notch1 Receptor Human genes 0.000 description 3
- 241000242739 Renilla Species 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 230000008045 co-localization Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 206010012818 diffuse large B-cell lymphoma Diseases 0.000 description 3
- 230000008482 dysregulation Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 238000012744 immunostaining Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000010232 migration assay Methods 0.000 description 3
- 229940028444 muse Drugs 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 3
- GMVPRGQOIOIIMI-DWKJAMRDSA-N prostaglandin E1 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1CCCCCCC(O)=O GMVPRGQOIOIIMI-DWKJAMRDSA-N 0.000 description 3
- 230000004850 protein–protein interaction Effects 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 206010062113 splenic marginal zone lymphoma Diseases 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 238000007482 whole exome sequencing Methods 0.000 description 3
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 2
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 2
- 208000011691 Burkitt lymphomas Diseases 0.000 description 2
- 102000003952 Caspase 3 Human genes 0.000 description 2
- 108090000397 Caspase 3 Proteins 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 102100031673 Corneodesmosin Human genes 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108090000331 Firefly luciferases Proteins 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101000952934 Homo sapiens Atrial natriuretic peptide-converting enzyme Proteins 0.000 description 2
- 101000847538 Homo sapiens Flotillin-1 Proteins 0.000 description 2
- 101001043598 Homo sapiens Low-density lipoprotein receptor-related protein 4 Proteins 0.000 description 2
- 101000666934 Homo sapiens Very low-density lipoprotein receptor Proteins 0.000 description 2
- 108010001831 LDL receptors Proteins 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 102100024640 Low-density lipoprotein receptor Human genes 0.000 description 2
- 102100021918 Low-density lipoprotein receptor-related protein 4 Human genes 0.000 description 2
- 208000034578 Multiple myelomas Diseases 0.000 description 2
- 230000005913 Notch signaling pathway Effects 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 102100021923 Prolow-density lipoprotein receptor-related protein 1 Human genes 0.000 description 2
- 108010029485 Protein Isoforms Proteins 0.000 description 2
- 102000001708 Protein Isoforms Human genes 0.000 description 2
- 102100029753 Reduced folate transporter Human genes 0.000 description 2
- 108010052090 Renilla Luciferases Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 102100039066 Very low-density lipoprotein receptor Human genes 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000002843 lactate dehydrogenase assay Methods 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 230000017448 oviposition Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000000306 recurrent effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 230000005740 tumor formation Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 210000003905 vulva Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 125000003821 2-(trimethylsilyl)ethoxymethyl group Chemical group [H]C([H])([H])[Si](C([H])([H])[H])(C([H])([H])[H])C([H])([H])C(OC([H])([H])[*])([H])[H] 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 206010073478 Anaplastic large-cell lymphoma Diseases 0.000 description 1
- 108050005848 Annexin A10 Proteins 0.000 description 1
- 241001156002 Anthonomus pomorum Species 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000167854 Bourreria succulenta Species 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- 241000244202 Caenorhabditis Species 0.000 description 1
- 241000244203 Caenorhabditis elegans Species 0.000 description 1
- 101100337060 Caenorhabditis elegans glp-1 gene Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102100035888 Caveolin-1 Human genes 0.000 description 1
- 238000007808 Cell invasion assay Methods 0.000 description 1
- 102100038215 Chromodomain-helicase-DNA-binding protein 7 Human genes 0.000 description 1
- 108010039419 Connective Tissue Growth Factor Proteins 0.000 description 1
- 102000015225 Connective Tissue Growth Factor Human genes 0.000 description 1
- 101710139375 Corneodesmosin Proteins 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- 102100037147 Cytoplasmic dynein 2 heavy chain 1 Human genes 0.000 description 1
- 102100036462 Delta-like protein 1 Human genes 0.000 description 1
- 241000586568 Diaspidiotus perniciosus Species 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 241000611421 Elia Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241001131798 Escherichia coli HT115 Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102100031510 Fibrillin-2 Human genes 0.000 description 1
- 101150103317 GAL80 gene Proteins 0.000 description 1
- 108010001515 Galectin 4 Proteins 0.000 description 1
- 102100039556 Galectin-4 Human genes 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 230000004655 Hippo pathway Effects 0.000 description 1
- 101000715467 Homo sapiens Caveolin-1 Proteins 0.000 description 1
- 101000943088 Homo sapiens Choline dehydrogenase, mitochondrial Proteins 0.000 description 1
- 101000883739 Homo sapiens Chromodomain-helicase-DNA-binding protein 7 Proteins 0.000 description 1
- 101000881344 Homo sapiens Cytoplasmic dynein 2 heavy chain 1 Proteins 0.000 description 1
- 101000928537 Homo sapiens Delta-like protein 1 Proteins 0.000 description 1
- 101000846890 Homo sapiens Fibrillin-2 Proteins 0.000 description 1
- 101001039207 Homo sapiens Low-density lipoprotein receptor-related protein 8 Proteins 0.000 description 1
- 101000663006 Homo sapiens Poly [ADP-ribose] polymerase tankyrase-1 Proteins 0.000 description 1
- 101000843572 Homo sapiens Transcription factor HES-2 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 108010003718 LDL-Receptor Related Protein-Associated Protein Proteins 0.000 description 1
- 208000032004 Large-Cell Anaplastic Lymphoma Diseases 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108010015340 Low Density Lipoprotein Receptor-Related Protein-1 Proteins 0.000 description 1
- 101710172064 Low-density lipoprotein receptor-related protein Proteins 0.000 description 1
- 102100040705 Low-density lipoprotein receptor-related protein 8 Human genes 0.000 description 1
- 206010064912 Malignant transformation Diseases 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 235000006679 Mentha X verticillata Nutrition 0.000 description 1
- 235000002899 Mentha suaveolens Nutrition 0.000 description 1
- 235000001636 Mentha x rotundifolia Nutrition 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 101000943091 Mus musculus Choline dehydrogenase, mitochondrial Proteins 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 102400000552 Notch 1 intracellular domain Human genes 0.000 description 1
- 101800001628 Notch 1 intracellular domain Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010051742 Platelet-Derived Growth Factor beta Receptor Proteins 0.000 description 1
- 102000018967 Platelet-Derived Growth Factor beta Receptor Human genes 0.000 description 1
- 229920002873 Polyethylenimine Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 101710202113 Prolow-density lipoprotein receptor-related protein 1 Proteins 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 102000002727 Protein Tyrosine Phosphatase Human genes 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 206010041660 Splenomegaly Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 206010042971 T-cell lymphoma Diseases 0.000 description 1
- 208000027585 T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 102100030772 Transcription factor HES-2 Human genes 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 108010031318 Vitronectin Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 201000011529 cardiovascular cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 235000019693 cherries Nutrition 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000000749 co-immunoprecipitation Methods 0.000 description 1
- 238000001360 collision-induced dissociation Methods 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 210000003674 cytoplasmic vesicle Anatomy 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000005350 fused silica glass Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000003540 gamma secretase inhibitor Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 102000056825 human TNKS Human genes 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000004966 intestinal stem cell Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 101150063692 lin-12 gene Proteins 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000004777 loss-of-function mutation Effects 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 238000003468 luciferase reporter gene assay Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000036212 malign transformation Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 1
- 229960003105 metformin Drugs 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 210000001178 neural stem cell Anatomy 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000011597 peroxisome degradation Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920000333 poly(propyleneimine) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 108020000494 protein-tyrosine phosphatase Proteins 0.000 description 1
- 238000000575 proteomic method Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 210000001350 reed-sternberg cell Anatomy 0.000 description 1
- 230000012760 regulation of cell migration Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 101150081985 scrib gene Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 102000035025 signaling receptors Human genes 0.000 description 1
- 108091005475 signaling receptors Proteins 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- IBKZNJXGCYVTBZ-IDBHZBAZSA-M sodium;1-[3-[2-[5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyldisulfanyl]propanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCSSCCNC(=O)CCCC[C@H]1[C@H]2NC(=O)N[C@H]2CS1 IBKZNJXGCYVTBZ-IDBHZBAZSA-M 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 108010051423 streptavidin-agarose Proteins 0.000 description 1
- 108010018381 streptavidin-binding peptide Proteins 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000003956 synaptic plasticity Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 102000013498 tau Proteins Human genes 0.000 description 1
- 108010026424 tau Proteins Proteins 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000008728 vascular permeability Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000012049 whole transcriptome sequencing Methods 0.000 description 1
- 239000012130 whole-cell lysate Substances 0.000 description 1
- 238000012447 xenograft mouse model Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Definitions
- the present disclosure generally relates to a method for the down-regulation of LRP1 using related inhibitors so as to treat Notch signaling-dependent disease.
- Notch signaling is highly conserved in various species ranging from Caenorhabditis elegans (C. elegans) to mammals and is regarded as one of the most important signaling pathways. Dysregulation of Notch signaling has been linked to multiple human disorders ranging from developmental syndromes to complex diseases such as Alzheimer’s disease, cardiovascular diseases, and cancers. Activating mutations of Notch 1/2 have been uncovered in patients with T-cell acute lymphoblastic leukemia (T-ALL) , chronic lymphocytic leukemia (CLL) , and many other types of cancers, while loss-of-function mutations in Notch receptors have been identified in patients with several squamous cell carcinomas (SCC) .
- T-ALL T-cell acute lymphoblastic leukemia
- CLL chronic lymphocytic leukemia
- SCC squamous cell carcinomas
- LRP1 is a large multiligand endocytic receptor that belongs to the low-density lipoprotein receptor family. Members of this family have been reported to be involved in cholesterol metabolism, intracellular trafficking and cell signal transduction, as well as in the regulation of cell migration, proliferation, synaptic plasticity, neuron development and cerebral vascular permeability maintenance.
- LRP1 is synthesized as a 600-kDa precursor protein and then processed into an extracellular ligand-binding subunit of 515 kDa (LRP1 ⁇ ) and a transmembrane and intracellular subunit of 85 kDa (LRP1 ⁇ ) , which is associated with efficient endocytic trafficking and intracellular signal transduction.
- LRP1 As an endocytic receptor, LRP1 promotes the internalization of many extracellular ligands, such as PDGFR- ⁇ , amyloid- ⁇ , Tau and CCN2, through endocytosis and transfers them to endosomes and lysosomal complexes. Recent studies have uncovered that LRP1 is expressed by neural stem cells, acts as a critical regulator of oligodendrocyte progenitor cells behavior and early astroglial differentiation and involved in their differentiation, further indicating its participation in the Notch pathway, as Notch pathway is central in radial glia differentiation.
- the present disclosure provides down-regulation of LRP1 using genetic means or related inhibitors can facilitate Notch signaling inhibition, so as to reduce leukemia cell invasion, migration, anchorage-independent cell growth and tumorigenesis. Therefore, the disclosure reveals the essential role of LRP1 in Notch signaling dependent disease, and provides a novel strategy for treating Notch signaling dependent disease such as T-cell acute lymphoblastic leukemia (T-ALL) ; meanwhile, the invention provides novel medicines for treating Notch signaling dependent disease, and further points out a new direction for screening medicine and therapeutic target for the treating Notch signaling dependent disease.
- T-ALL T-cell acute lymphoblastic leukemia
- the disclosure relates to a novel method of treating Notch signaling-dependent disease.
- the present disclosure provides a method for treating Notch signaling-dependent disease by using a LRP1 inhibitor, which may be a polypeptide antagonist specifically against LRP1, an RNA polynucleotide specific to LRP1, or a small molecule compound inhibitor specific to LRP1.
- the invention provides a LRP1 specific inhibitor for use in treating Notch signaling-dependent disease.
- the LRP1 inhibitor is selected from a polypeptide antagonist specifically against LRP1, an RNA polynucleotide specific to LRP1, or a small molecule compound inhibitor specific to LRP1.
- the invention provides use of a LRP1 specific inhibitor in preparation of medicine for treating Notch signaling-dependent disease.
- the LRP1 inhibitor is a polypeptide antagonist specifically against LRP1, an RNA polynucleotide specific to LRP1, or a small molecule compound inhibitor specific to LRP1
- polypeptide antagonist is LRPAP1 or LRPAP1 derivative thereof that can bind to LRP1 on the cell surface and prevent ligands from its binding.
- the polypeptide antagonist is selected from LRPAP1 comprising an amino sequence of SEQ ID NO: 1 or 2, an amino acid sequence with at least 70%, 80%, 85%, 90%, 95%, 99%, or more identity to SEQ ID NO: 1 or 2, or an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 1 or 2. and the LRPAP1 can bind to LRP1 on the cell surface, preventing ligands from its binding.
- LRPAP1 derivative is a polypeptide comprising: an amino acid sequence of SEQ ID NO: 3; an amino acid sequence an amino acid sequence with at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity to SEQ ID NO: 3; or an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 3; and the LRPAP1 derivatives can bind to LRP1 on the cell surface, preventing ligands from its binding.
- LRPAP1 derivative is a polypeptide comprising: an amino acid sequence of SEQ ID NO: 4; an amino acid sequence an amino acid sequence with at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity to SEQ ID NO: 4; or an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 4; and the LRPAP1 derivatives can bind to LRP1 on the cell surface, preventing ligands from its binding.
- the LRPAP1 derivative is a polypeptide comprising SEQ ID NO: 4 (RAPm6) .
- the LRPAP1 or LRPAP1 derivative is a polypeptide without or with a tag at the C-terminal of N-terminal of any sequence of SEQ ID NO: 1-4.
- the tag is selected from c-Myc, His, HA, GST, MBP, Flag, and Arg6.
- LRPAP1 derivative is a polypeptide of SEQ ID NO: 4.
- the LRPAP1 or LRPAP1 derivative is a polypeptide modified by PEG.
- the polypeptide antagonist is an antibody against LRP1.
- the RNA polynucleotide is selected from siRNA, shRNA, guide RNA, and miRNA.
- the guide RNA is SEQ ID NO: 5 (TGGAGGACAAGATCTACCGC) .
- the Notch signaling-dependent disease is selected from leukemia e.g. T-acute lymphoblastic leukemia or Chronic lymphocytic leukemia, myeloma e.g. Multiple myeloma, lymphoma e.g. Hodgkin lymphoma, Burkitt lymphoma, Diffuse large B-cell lymphoma, Mantle cell lymphoma, Splenic marginal zone lymphoma, Follicular lymphoma, breast cancer, liver cancer, lung cancer, and lung adenocarcinoma cells.
- the leukemia is T-acute lymphoblastic leukemia or Chronic lymphocytic leukemia.
- the disease is any type of leukemia.
- the subject is non-human mammal or human.
- the invention provides a method of screening medicines for treating Notch signaling-dependent disease using LRP1 as the target, the method comprising: observing the effect of candidate medicine on the expression or activity level of LRP1, if the candidate medicine can inhibit expression or activity level of LRP1, then it indicates that the candidate medicine is a potential medicine for treating Notch signaling-dependent disease.
- the Notch signaling-dependent disease is selected from leukemia, The leukemia is selected from acute lymphoblastic leukemia (ALL) , chronic lymphocytic leukemia (CLL) .
- Fig. 1. shows the expression of LRP1 gene in the control group and leukemia patients was evaluated by sequencing.
- the leukemia patients and healthy individuals are represented by boxes, respectively.
- FIG. 2 shows LRP1 directly interacts with DLL3 and promotes its membrane localization and stability.
- A-H LRP1 ⁇ interacts with DLL3 in cells and in vitro.
- A, B HEK293T cell lysates were incubated with IgG control and antibodies recognizing DLL3 (A) or LRP1 ⁇ or LRP1 ⁇ (B) . Five percent lysate was used as the input control. Blots with antibodies recognizing actin, DLL3, LRP1 ⁇ or LRP1 ⁇ are shown.
- C-F Mapping the binding regions between LRP1 ⁇ and DLL3.
- C, D Schematics of LRP1 ⁇ (C) and DLL3 (D) domain deletion mutants for domain mapping assays.
- E, F HEK293T cells were cotransfected with (E) Myc-tagged DLL3 and cSFB-tagged wild-type or mutant LRP1 ⁇ or (F) Myc-tagged LRP1 ⁇ and cSFB-tagged wild-type or mutant DLL3. The cell lysates were incubated with S beads. Five percent lysate was used as the input control. Blots with antibodies recognizing the FLAG and MYC epitope tags and actin are shown. (G) Colocalization assay of LRP1 and DLL3.
- HEK293T cells were transfected with cSFB-LRP1 ⁇ and Myc-DLL3 and subjected to immunofluorescence with an anti-Myc antibody against DLL3 (red) , an anti-Flag antibody against LRP1 ⁇ (green) and DAPI (blue) and visualized by microscopy. Scale bars, 10 ⁇ m. Quantitation of immunofluorescence colocalization of LRP1 ⁇ and DLL3, Pearson's R value was calculated with ImageJ software, and Pearson's R value >0.5 was considered as good colocalization. (H) In vitro GST pull-down assay of LRP1 ⁇ and DLL3.
- Wild-type or LRP1-KO HEK293T cells were subjected to western blotting with antibodies recognizing DLL3, LRP1 ⁇ , NOTCH1 and actin.
- K Knocking out LRP1 had no prominent effect on NOTCH1 localization. Wild-type or LRP1-KO HEK293T cells were subjected to immunofluorescence with an anti-NOTCH1 antibody (green) and DAPI (blue) and visualized by microscopy. Scale bars, 10 ⁇ m.
- (L) Wild-type or LRP1-KO HEK293T cells were homogenized and separated into cytosolic and membrane fractions.
- Lysates were subjected to western blotting with antibodies recognizing DLL3, LRP1 ⁇ , CAV1 and actin.
- M, N Knocking out LRP1 impairs Notch signaling.
- M The mRNA levels of LRP1 and the Notch pathway target genes in wild-type and LRP1-KO HEK293T cells were determined by RT-qPCR.
- N Wild-type or LRP1-KO HEK293T cells were cotransfected with HES1 or HES5 luciferase constructs, respectively, and a Renilla luciferase construct.
- Figure 3 shows LRP1 homologues function as important Notch pathway upstream regulators in C. elegans and Drosophila.
- A-E Overexpression of Notch signaling receptor LIN-12 reverses the molting defect induced by lrp-1 RNAi in C. elegans.
- A Experimental workflow for examining the effects of lrp-1 RNAi in WT and lin-12 overexpression lines.
- B Molting defects induced by lrp-1 RNAi in WT worms were visualized by microscopy. All phenotypes correlated percentages was collected and presented, respectively.
- C RNAi efficiencies for both lines.
- RNAi downregulation of dlg induced Dl upregulation (I’) , invasion cell migration (I”) , and MMP1 induction (O’) were all suppressed by reducing LRP1 activity (J’, J” and P’) .
- K-M’ LRP1 knockdown in the posterior region of wing discs reduces endogenous Cut expression.
- Q-R Fluorescence micrographs of posterior adult midgut were shown. 8 days of LRP1 knockdown in the ISCs increased ISC proliferation (Cherry positive) and EE number (Pros positive) .
- S Quantification of relative Pros+ cells in Q” and R”.
- T Quantification of PH3+ mitotic cells per gut in Q’” and R’”.
- FIG. 4 shows that knocking out LRP1 attenuates Notch signaling-dependent leukemia invasion, migration, and tumorigenesis.
- A, B LRP1 is overexpressed in leukemia cell lines.
- A Western blots with antibodies recognizing LRP1 ⁇ and actin in various leukemia cell lines.
- B, C The protein levels of DLL3 and NOTCH1 were determined in wild-type and LRP1-KO HSB2 (B) or K562 (C) cells by western blotting.
- D Knocking out LRP1 attenuates the expression of Notch target genes. The mRNA levels of the Notch target genes in wild-type and LRP1-KO HSB2 cells were determined by RT-qPCR.
- J, L Migration abilities of wild-type and LRP1-KO HSB2 (J) or K562 (L) cells were measured using a transwell migration assay. Scale bars, 100 ⁇ m.
- K, M The numbers of cells that migrated into the lower chamber in J and L were counted.
- N, P Anchorage-independent tumorigenesis abilities of wild-type and LRP1-KO HSB2 (N) or K562 (P) cells were measured using a soft agar colony formation assay.
- O, Q The numbers of colonies in N, P, were counted.
- R Xenograft tumor growth studies were performed using wild-type or LRP1-KO HSB2 cells.
- mice were euthanized after 4 weeks of injection.
- the tumors were excised, photographed, and weighed.
- S, T The volumes (S) and weights (T) of the tumors were measured.
- U The levels of DLL3 protein in tumors in (R) were detected by western blotting. Blots with antibodies recognizing DLL3 and actin are shown.
- V Knocking out LRP1 attenuates the expression of Notch target genes.
- the mRNA levels of the Notch target genes in tumors derived from wild-type and LRP1-KO HSB2 cells were determined by RT-qPCR.
- the data are shown as the mean ⁇ SEM from the indicated numbers of independent experiments. P values were calculated using two-tailed Student’s t-tests (*P ⁇ 0.05, **P ⁇ 0.01, NS, not significant) .
- Figure 5 shows that comparison of WT and LRP1-KO cells from multiple perspectives.
- A The viability of WT and LRP1-KO HEK293T cells was measured using LDH release assay, Triton X-100 was used as a positive control.
- B-D The influence of LRP1 KO on cell apoptosis in HEK293T, HSB2 and K562 cells was evaluated via Caspase3 level detection (B) and Annexin V/PI staining (C-D) .
- E The lipid raft was isolated from both WT and LRP1-KO HEK293T cells and evaluated via western blotting using FLOT1 as lipid raft marker.
- FIG. 6 shows that Overexpression of NICD1 rescues LRP1-KO phenotypes in leukemia cells but not in MDA-MB-231 cells.
- A, C, H Anchorage-independent tumorigenesis abilities of LRP1-KO and LRP1-KO+NICD1 HSB2 (A) , K562 (C) or MDA-MB-231 (H) cells were measured using a soft agar colony formation assay.
- (M) Anchorage-independent tumorigenesis abilities of MDA-MB-231 breast cancer cells treated with the vehicle or 0.1 mg/mL GST-RAPm6 for 36 h were measured using a soft agar colony formation assay.
- FIG. 7 shows that LRP1 antagonist RAPm6 inhibits tumorigenesis in human leukemia cells, mouse xenografts and leukemia models.
- A-C RAPm6 interacts with LRP1 and inhibits Notch signaling.
- A GST and GST-RAPm6 were expressed and purified in E. coli and subjected to SDS-PAGE followed by Coomassie bright blue staining.
- B In vitro GST pull-down assay of LRP1 ⁇ and RAPm6. HEK293T cell lysates were coincubated with GST or GST-RAPm6 and glutathione sepharose for 2 h. Five percent lysate was used as the input control.
- the pull-down assays were subjected to SDS-PAGE followed by western blotting with antibodies recognizing LRP1 ⁇ and actin.
- C HSB2 cells were treated with the vehicle or RAPm6. The mRNA levels of LRP1 and Notch pathway target genes were determined by RT-qPCR.
- D-Q RAPm6 inhibits cell viability (D) and tumorigenesis in human leukemia cells (E-H) , mouse xenografts (I-L) and leukemia (M-Q) models.
- D HSB2, K562 and DND41 leukemia cells were treated with 0.1 mg/mL GST-RAPm6 for 36 h. Cell viabilities were analyzed using a CCK-8 assay.
- E, G Anchorage-independent tumorigenesis abilities of wild-type and LRP1-KO HSB2 (E) or K562 (G) cells were measured using a soft agar colony formation assay.
- F, H The numbers of colonies in E, G, were counted.
- I Xenograft tumor growth studies were performed using HSB2 cells. Mice were treated with the vehicle or GST-RAPm6 and euthanized 4 weeks after injection. The tumors were excised, photographed, and weighed.
- J, K The volumes (J) and weights (K) of the tumors were measured.
- L The weights of the mice were measured.
- mice Mouse leukemia models were established in NOD-SCID mice by injecting PBS or HSB2 cells intravenously via the tail vein. Mice were treated with the vehicle or GST-RAPm6. The percentages of CD5+ leukemia cells in peripheral blood were measured weekly by flow cytometry analysis.
- O The survival time of mice in m was recorded.
- P Spleens from mice in m were excised, and representative pictures of each group are shown.
- Q Representative pictures of HE-stained spleens from P. Scale bars, 50 ⁇ m.
- a polypeptide means one polypeptide or more than one polypeptide.
- LRP1 used herein refers to proteins encoded by low density lipoprotein receptor-related protein LRP1 gene in C. elegans, Drosophila, or mammals. LRP1 gene.
- Term “inhibitor” used herein refers to materials capable of lowering, reducing or eliminating the amount, particular function, and particular property of a target object.
- Said target object can be a protein, polypeptide, nucleic acid and the like, while said inhibitor affects the amount, particular function, and particular property of the target object either directly or indirectly so as to result in the corresponding lowering, reducing or eliminating of the amount, particular function, and particular property of the target object.
- Said inhibitor can be a protein, polypeptide, nucleic acid, small molecule compound and the like.
- LRP1 inhibitor refers to materials capable of lowering, reducing or eliminating the expression, transcription, translation of LRP1 gene, and/or stability of LRP1 protein produced therefrom, binding ability to protein etc., which includes but is not limited to a polypeptide antagonist against LRP1, inhibitory nucleotides specific to LRP1, antibodies against LRP1 protein, small molecule compound inhibitors capable of inhibiting LRP1 activity, and/or materials capable of inhibiting the interaction between LRP1 protein and other membrane proteins, and the like.
- LRPAP1 refers to an antagonist of low-density lipoprotein receptor-related protein 1 (LRP1) .
- LRPAP1 can bind to LRP1 on the cell surface, preventing ligands from binding.
- binding or “binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between a ligand and a receptor.
- LRPAP1 can be polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or 2 or an amino acid sequence at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity to SEQ ID NO: 1 or 2, or an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 1 or 2.
- LRPAP1 derivatives refers to a truncated LRPAP1 or its mutation and the polypeptide can bind to LRP1 on the cell surface, preventing ligands from binding.
- LRPAP1 derivatives may be a truncated LRPAP1 shown as an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence an amino acid sequence with at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity to SEQ ID NO: 2, or an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 2.
- LRPAP1 derivatives can also bind to LRP1 on the cell surface, preventing ligands from binding.
- LRPAP1 derivative can be RAPm6 shown as an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence with at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity to SEQ ID NO: 4, or an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 4.
- antibody used herein refers to any immunoglobulin or complete molecule and fragments thereof which binds to a specific epitope. Said antibody includes but not limited to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, and fragments and/or parts of intact antibodies, as long as such fragments or parts retain the antigen binding capacity of the parent antibody.
- antibody against LRP1 refers to monoclonal antibodies, polyclonal antibodies, single chain antibodies and immunological activie fragments or parts thereof capable of specific binding to LRP1 protein, or functional variants or functional fragments thereof.
- terms such as “LRP1 antibody” , “antibody against LRP1” , and “anti-LRP1 antibody” are used interchangeably.
- “functional variant” refers to the protein or polypeptide of the invention with one or more amino acid modification in its amino acid sequence.
- the modification can be a "conservative” modification (wherein the substituted amino acid has similar structure or chemical property) or a “non-conservative” modification; similar modification also include addition or deletion of amino acid or both.
- conservative modification wherein the substituted amino acid has similar structure or chemical property
- non-conservative modification also include addition or deletion of amino acid or both.
- neither the modification of amino acid residue nor the addition or deletion of amino acid would substaintially change or damage the biological or immunological activity and function of the original amino acid sequence.
- “functional fragment” refers to any part of the protein or polypeptide of the invention, which retains the substantially similar or identical biological or immunological activity and function of the protein or polypeptide of which it is a part (the parent protein or polypeptide) .
- RNA polynucleotide specific to LRP1 refers to nucleotide capable of binding to and/or inhibiting expression of LRP1 gene.
- Typical inhibitory nucleotide includes but not limited to antisense oligonucleotides, triple helix DNAs, RNA aptamers, ribozymes, small interfering RNA (siRNA) , short hairpin RNA (shRNA) and microRNA. These nucleotide compounds bind to said specific genes with higher affinity than other nucleotide sequences, so as to inhibit expression of the specific genes.
- Term "small molecule compound” used herein refers to organic compounds with molecular weight less than 3k dalton which can be either natural or chemically synthesized.
- Term “derivative” used herein refers to compounds generated by modifying the parent organic compound through one or more chemical reactions, which have similar structures as the parent organic compound and similar effects in their functions.
- Term “analogue” used herein refers to compounds which were not generated by chemically modifying the parent organic compound but are similar to the parent organic compound in structure and have similar effects in their functions.
- Term “disease” used herein refers to Notch signaling dependent disease e.g. Notch signaling acitivated cancers.
- the cancer can be but not limited the T-acute lymphoblastic leukemia (Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science. 2004; 306: 269-71.
- CUTLL1 a novel human T-cell lymphoma cell line with t (7;9) rearrangement, aberrant NOTCH1 activation and high sensitivity to gamma-secretase inhibitors.
- Leukemia. 2006; 20: 1279-87 Chronic lymphocytic leukemia (NOTCH1 mutations influence survival in chronic lymphocytic leukemia patients.
- BMC Cancer Chronic lymphocytic leukemia
- lymphoma e.g. Hodgkin lymphoma (Activated Notch1 signaling promotes tumor cell proliferation and survival in Hodgkin and anaplastic large cell lymphoma. Blood. 2002; 99: 3398-403. ) , Burkitt lymphoma (Notch is an essential upstream regulator of NF-kappaB and is relevant for survival of Hodgkin and Reed-Sternberg cells. Leukemia.
- lung adenocarcinoma cells (Notch-1 stimulates survival of lung adenocarcinoma cells during hypoxia by activating the IGF-1R pathway. Oncogene 29, 2488–2498 (2010) . Oxygen concentration determines the biological effects of NOTCH-1 signaling in adenocarcinoma of the lung. Cancer Res. 67, 7954–7959 (2007) . ) .
- Term "therapeutic target” used herein refers to various materials that can be used to treat a certain disease and the target of the material in animal or human bodies. Treatment effects on said disease are obtainable when said materials act on said target.
- Said materials can be a variety of materials such as protein, polypeptide, nucleic acid, small molecule compound, said target can be material substances such as a certain gene (including a specific sequence of a gene) , a ceratin protein (including a specific site of a protein) , a certain protein complex (including specific binding site thereof) , or certain charactistics, certain functions, certain interaction relationships with peripheral substances and environment of aforementioned genes and/or proteins, etc, as long as said materials can affect the gene, protein, protein complex, or charactistic, function, interaction relationship thereof so as to treat the disease.
- the term “subject” includes any human or nonhuman animal.
- nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms “patient” or “subject” are used interchangeably.
- treat refers to reversing, ameliorating or inhibiting the progression of the disease to which the term is applied, or one or more symptoms of the disease.
- the term also include prevention of disease, which includes the prevention of disease or the onset of any symptoms associated therewith, and ameliorating symptoms or reducing the severity of any condition before its onset.
- Percent (%) sequence identity with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F.
- HEK293T and MDA-MB-231 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Australia) supplemented with 10%fetal bovine serum.
- DMEM Dulbecco's modified Eagle's medium
- JURKAT, K562, DND-41, ICHIKAWA and HSB2 cells were cultured in RPMI 1640 medium supplemented with 10%fetal bovine serum. All culture media contained 10%fetal bovine serum (FBS, Gibco, Australia) and were supplemented with 1%penicillin and streptomycin (Thermo Fisher Scientific, USA) .
- cDNAs encoding all of the genes were obtained from the hORFV5.1 library or amplified from cDNA of HEK293T cells using RT-PCR. cDNAs were subcloned into the pDONR201 vector (Invitrogen, USA) as entry clones and subsequently transferred to Gateway-compatible destination vectors for the expression of C-terminal streptavidin-binding peptide (SFB) triple tagged- (S protein tag-2 ⁇ FLAG tag-SBP tag) or MYC-tagged fusion proteins. Deletion mutants of LRP1 and DLL3 were generated using site-directed mutagenesis. sgRNA against LRP1 was synthesized and cloned into the pLenti-V2 vector (Addgene #52961) . All constructs were confirmed by sequencing.
- sgRNA constructs were packaged into lentiviruses by co-transfecting them with the packaging plasmids pMD2G and pSPAX2 into HEK293T cells. Forty-eight hours after transfection, the supernatant was collected and used to infect HEK293T, MDA-MB-231, HSB2 or K562 cells. Infections were repeated twice at an interval of 24 h to achieve maximal infection efficiency. Stable cells were selected using medium containing 2-5 ⁇ g/mL puromycin. Overexpression or knocking out efficiencies were confirmed using Western blot analysis.
- the membrane-bound and soluble proteins of 2 ⁇ 10 8 HEK293T cells were extracted using a membrane/soluble protein isolation kit (Beyotime, China) with protease inhibitors at 4°C.
- the insoluble pellets from the crude lysis step were briefly sonicated and incubated with TurboNuclease for 30 min at 37°C with occasional vortexing to extract chromatin-bound protein complexes.
- the lysates were then centrifuged at 14,000 rpm for 30 min at 4°C, and the supernatant was collected as a chromatin fraction. All three fractions were combined and incubated with streptavidin-conjugated beads (GE, USA) for 2 h at 4°C.
- streptavidin-conjugated beads GE, USA
- the beads were washed three times with NETN buffer, and bound proteins were eluted with NETN buffer containing 2 mg/mL biotin (Sigma, USA) for 2 h at 4°C. The elutes were incubated with S-protein beads (EMD Millipore, USA) for 1 h. The beads were washed three times with NETN buffer and subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) . Each pull-down sample was run just in the separation gel so that the whole band could be excised as one sample and subjected to in-gel trypsin digestion and LC-MS.
- SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis
- a nanoscale reverse-phase high-performance liquid chromatography capillary column was created by packing 5- ⁇ m C18 spherical silica beads into a fused silica capillary (100 ⁇ m inner diameter ⁇ ⁇ 20 cm length) using a flame-drawn tip. After the column was equilibrated, each sample was loaded onto the column using an autosampler. A gradient was formed, and peptides were eluted with increasing concentrations of solvent B (97.5%acetonitrile and 0.1%formic acid) .
- peptides eluted they were subjected to electrospray ionization and then analyzed by an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific, USA) .
- the source was operated at 1.9 kV, with no sheath gas flow and with the ion transfer tube at 350°C.
- the data-dependent acquisition mode was used.
- the survey scan was conducted from m/z 350 to 1, 500, with a resolution of 60,000 at m/z 200.
- the 20 most intense peaks with charge states of 2 and greater were acquired with collision-induced dissociation with a normalized collision energy of 30%and one micro scan; the intensity threshold was set at 1,000.
- MS2 spectra were acquired with a resolution of 15,000.
- the peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide.
- Peptide sequences and, hence, protein identity was determined by matching fragmentation patterns in protein databases using the Mascot software program (Matrix Science, USA) .
- Enzyme specificity was set to partially tryptic with two missed cleavages.
- Modifications of the peptides included carboxyamidomethyl (cysteines, variable) and oxidation (methionine, variable) .
- Mass tolerance was set to 20 ppm for both precursor ions and fragment ions.
- the database searched was Swiss-Prot (Homo sapiens) .
- Spectral matches were filtered to contain the false-discovery rate to less than 1%at the peptide level using the target-decoy method (Elias and Gygi, 2007) , and protein inference was considered following the general rules (Nesvizhskii and Aebersold, 2005) with manual annotation applied when necessary. This same principle was used for protein isoforms when they were present. Generally, the longest isoform was reported.
- MS data analysis was performed using the MUSE algorithm as described previously to assign quality scores for the identified PPIs. Twenty-two unrelated TAP-MS experiments using overexpressed TAP-tagged protein baits performed under identical experimental conditions were used as controls for the MUSE analysis. A MUSE score was assigned to each identified interaction, and any interaction with a MUSE score of at least 0.85 and raw spectral counts greater than 1 was considered to be an HCIP.
- P values were estimated using the Knowledge Base included with the Ingenuity Pathway Analysis software program (Ingenuity Systems, USA) , which contained findings and annotations from multiple sources, including the Gene Ontology, KEGG pathway, and PANTHER Pathway databases. Only statistically significant correlations (P ⁇ 0.05) are shown. The -log (P value) for each function and related HCIPs is listed.
- CHD patient data sets were downloaded from previous study (Jin, S.C., Homsy, J., Zaidi, S., Lu, Q., Morton, S., DePalma, S.R., Zeng, X., Qi, H., Chang, W., Sierant, M.C., et al. (2017) . Contribution of rare inherited and de novo variants in 2,871 congenital heart disease probands. Nat Genet 49, 1593-1601. ) . Cancer patient data sets were downloaded from cBioPortal and an exome sequencing data of 130 T-ALL patients.
- NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5%Nonidet P-40) on ice for 30 min and then boiling them in 2 ⁇ Laemmli buffer.
- NETN buffer 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5%Nonidet P-40
- we treated insoluble pellets resulting from crude lysis with TurboNuclease (Accelagen) which hydrolyses single-and double-stranded DNA and RNA to oligonucleotides that are 1-4 bases long to release chromatin-bound proteins (i.e., the chromatin fraction) .
- GST or GST-LRP1 ⁇ were first incubated with GST resin for 2 h in 4 °C, then purified SUMO-DLL3 protein was added after phosphate-buffered saline (PBS) wash three times and incubated for additional 2 h in 4 °C. Beads were washed three times with PBS and boiled in SDS loading buffer. Lysates were subjected to SDS-PAGE followed by Coomassie bright blue stain or WB.
- PBS phosphate-buffered saline
- cells were seeded in a cell culture dish, fixed with 4%paraformaldehyde at room temperature for 10 min. Cells were permeabilized 10 min with 0.1%TritonX-100, washed with PBS and blocked in 5%BSA in PBS for 30 minutes before labelling in primary antibodies at room temperature for 1h. and permeabilized at 4°Cfor 30 min.
- the cells were washed with PBS twice, stained with goat-anti-rabbit Fluorescein isothiocyanate-labelled IgG or goat-anti-mouse rhodamine-labelled IgG (1: 5000, Abcam, UK) at room temperature for 1 h, and subjected to 4’, 6-diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich, USA) .
- Coverslips were mounted using FluorSave TM Reagent (Milipore, USA) .
- the cells were viewed using an Olympus IX73 Microscope Imaging System (Olympus, Japan) .
- RNA from each sample was reverse-transcribed into cDNA using Highscript III reverse transcriptase (Vazyme, China) .
- Levels of mRNA for specific genes were quantified by qPCR using a Qtower3G qPCR system (Jena Bioscience, Germany) with SYBR Green Master Mix (Takara, Japan) . The data were normalized to the Actin expression level in each sample.
- E. coli HT115 was obtained from the Caenorhabditis Genetics Center, L4440 as empty vector.
- the lrp-1 RNAi clone from Ahringer library was used in this study and fed to C. elegans as described previously (Wu, L., Zhou, B., Oshiro-Rapley, N., Li, M., Paulo, J.A., Webster, C.M., Mou, F., Kacergis, M.C., Talkowski, M.E., Carr, C.E., et al. (2016) . An Ancient, Unified Mechanism for Metformin Growth Inhibition in C. elegans and Cancer. Cell 167, 1705-1718 e1713. ) .
- the P0 animals of N2 and WU45 were grown on L4440 bacteria from L4 stage. When reaching D2 adult stage, gravid animals were used for egg laying for 2 hours on L4440 and lrp-1 RNAi respectively. After 72 hours of RNAi feeding, the number of animals with molting defect were counted, analyzed relative to total worms, and imaged using a Leica DM500 microscope at a magnification of 10 ⁇ .
- the genomic sequence of lin-12 gene and 990 bp of the glp-1 promoter sequence were cloned into a plasmid vector pPD95.
- pPD95 plasmid vector
- mRFP monomeric red fluorescent protein
- Plasmid (20 ng/ ⁇ L) and injection marker myo-2p:: GFP (3 ng/ ⁇ l) were injected into the gonad of wild type adult animals and screened for the transgenic line according to fluorescence.
- RNAi lines were collected from the Vienna Drosophila Resource Center: UAS-LRP1. RNAi (v8397) , UAS-dlg. RNAi (v41136) . UAS-LRP1. RNAi (#2, THU3999) was obtained from Tsinghua Fly Center, Tsinghua University, Beijing China. esg-GAL4, UAS-mCherry, tub-Gal80ts; Su (H) Gbe-GAL80 was a gift from Hangsong Deng, Tongji University, Shanghai, China.
- Fluorescently labeled clones were produced in the eye discs by crossing FRT42D or FRT42D, LRP1EY07878 with the following strain: FRT42D, tub-Gal80; ey-Flp6, Act>y+>Gal4, UAS-GFP (42D tester) .
- Wing and eye imaginal discs of third-instar larvae were dissected in PBS and fixed in PBS containing 4%formaldehyde for 15 min, and fly intestines were fixed for 40 min.
- Flies used for gut dissection were reared at 18°C and 3-day-old adult female of the indicated genotypes were shifted to 29 °C to inactivate temperature sensitive GAL80 (GAL80ts) and allow expression of the transgenes for 8 or 14 days.
- GAL80ts temperature sensitive GAL80
- larvae were shift to 29°C one day after egg laying.
- RP49 was used as an internal control.
- the cholesterol uptake ability was evaluated using Cholesterol Uptake Assay Kit (ab236212, Abcam) according to the manufactures guidelines. Briefly, cells were seeded at a density of 5x10 5 cells/mL and incubated overnight in serum-free medium with 20 ⁇ g/mL NBD Cholesterol in a cell culture incubator at 37°C. The next day, culture medium was removed and replaced with an appropriate volume of assay buffer, then the degree of NBD cholesterol uptake was analyzed using a microplate reader.
- Trypan blue staining was used to determine the cell viability as follows. First, the cell suspension was prepared and then incubated with 0.4%Trypan Blue solution (T10282, Thermo Fisher Scientific) at 1: 1 ratio for 1-2 minutes at room temperature. Non-viable cells will be blue, viable cells will be unstained. Cell staining was observed under a light microscope and positively stained cell was calculated.
- 0.4%Trypan Blue solution T10282, Thermo Fisher Scientific
- Lactate dehydrogenase (LDH) assay To evaluate the integrity of cell membrane, the LDH release from these cell lines was measured using LDH Cytotoxicity Assay Kit (Beyotime) based on the manufacturer’s instruction. Briefly, both wild-type and LRP1-KO cell lines were collected and washed once with fresh regular culture medium, then seeded into a 96-well plate with 2-10 x 10 4 cells/well. After Incubating in an incubator (5 %CO2, 90 %humidity, 37°C) for the appropriate time of treatment, cells were centrifuged at 400 x g for 5 min to precipitate and the clear medium solution (120 ⁇ L/well) was transferred into an optically clear 96-well plate for detection.
- LDH Cytotoxicity Assay Kit Beyotime
- Membrane lipid raft was isolated using Minute TM Total Lipid Raft Isolation Kit (Invent Biotech) according to manufactures’ guidelines. 30-40 x 10 6 cells were collected by low speed centrifugation (500-600 x g for 5min) and washed once with cold PBS. Remove supernatant completely and resuspend the pellet in buffer A, incubating on ice for 5 min. Vortex the tube vigorously for 10-30 seconds. Immediately transfer the cell suspension to the filter cartridge. After a series of centrifugation, pellet containing total membrane fraction was acquired and resuspended in buffer B and C consecutively. Finally, the lipid rafts would adhere to the wall of the microfuge tube after removal of the aqueous phase and resuspended in 50-200 ⁇ L buffer for following western blotting analysis.
- Cell apoptosis was evaluated using Annexin V-FITC Apoptosis Detection Kit (Beyotime) according to the manufactures’ instructions. Briefly, 1-5 x10 5 cells were collected by centrifugation, followed by washing with cold 1X PBS and carefully remove the supernatant. Resuspend the cells in 195 ⁇ L Annexin V-FITC binding buffer and add 5 ⁇ L of Annexin V-FITC and 10 ⁇ L propidium iodide (PI) staining solution to tubes and gently swirl to mix. After incubating the mixture for 20 minutes at room temperature in the dark, cells were immediately analyzed by flowcytometry.
- Annexin V-FITC Apoptosis Detection Kit Beyotime
- the HES1 and HES5 promoter driven-luciferase reporter constructs were generated by insertion of the HES1 and HES5 promoter into pGL3-luc luciferase vector upstream of the firefly luciferase gene.
- luciferase assay cells were plated at 50%confluency in 24-well plate and grown overnight.
- the firefly luciferase reporter construct and the Renilla control reporter were contransfected into the cells at a molar ratio of 10: 1. After 24h of culture, the luciferase reporter activity was assayed with the Dual Luciferase Assay System (Promega) .
- WT and DLL3 KD cells were plated in a six-well plate overnight and then cotransfected with the luciferase reporter and Renilla control reporter as an internal control. Twenty-four hours after transfection, the transfected cells were cocultured with DLL1 overexpressed cells for additional 24 hr. In another case, WT cells were first cotransfected with the luciferase reporter and Renilla control reporter and then cocultured with WT and DLL3 KD cells respectively. Luciferase activities were measured using a Dual Luciferase Assay System (Promega) .
- Biotinylation-streptavidin pull down was performed essentially as described previously. Briefly, 293T cells expressing Myc-DLL3 were labeled on 4°C for 30 min with Sulfo-NHS-SS-Biotin solution (0.5 mg/mL in PBS) . After a 30 min incubation at 4°C or 37°C, biotin was stripped by twice 30 min incubation with 10 mM DTT in TNEB buffer (20 mM Tris, pH 8.3; 150 mM NaCl; 1 mM EDTA; 0.2%BSA) on ice. Cells were lysed with RIPA, biotinylated species were purified on streptavidin agarose and analyzed by immunoblotting with anti-Myc antibody
- Leukemia cells invasion was measured using a three-dimensional culture system with Matrigel (Corning, USA) .
- 5,000 wild-type or LRP1-KO HSB2 or K562 cells were mixed with 500 ⁇ L Matrigel and plated in 24-well plates.
- the spheres were visualized by microscopy 7 days post seeding.
- the numbers and the average diameters of spheres were measured using a GelDoc with Quantity One software (Bio-Rad, USA) .
- Leukemia cells migration was measured using a transwell migration assay. 50,000 wild-type or LRP1-KO HSB2 or K562 cells were seeded onto a 24-well transwell chamber. Cells migrated into the lower chamber were visualized by microscopy 36 h post seeding. The numbers of cells migrated into the lower chamber were counted manually.
- 3,000 wild-type or LRP1-KO HSB2 or K562 cells were added to 1.5 mL of growth medium with 1.4%agar and layered onto 2 mL of 2.4%agar bed in 6-well plates. Medium was replenished every week for 4 weeks. Resulting colonies were fixed and stained with 0.005%crystal violet solution overnight and photographed. The numbers of colonies were counted with a GelDoc with Quantity One software (Bio-Rad, USA) .
- mice All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the Westlake University.
- 5 ⁇ 10 6 each types of the cells eg. HSB2 vs HSB2 LRP1-KO
- PBS PBS
- Tumor formation was observed weekly and tumor sizes were measured. Mice were euthanized after 4 weeks of injection and the tumors were excised, photographed and weighed.
- Mouse leukemia models were established in NOD-SCID mice. 5 ⁇ 106 HSB2 cells were resuspended in 100 ⁇ L PBS and injected intravenously into 6-week-old female NOD-SCID mice via tail vein. Starting from the day 7, vehicle or RAPm6 protein was administered by tail vein injection for 3 days followed by 4 days of rest for a total of 4 cycles. After each cycle, peripheral blood leukemia cells were analyzed using flow cytometry as follows. Peripheral blood was collected from treated NOD-SCID mice and red blood cells were removed using RBC lysis (Beyotime, China) .
- mice After washing three times with PBS, cells were labelled in suspension with FITC mouse anti-human CD5 (BD Pharmingen, USA) for 30 min at 4 °C. Cells were then washed three times with PBS and analyzed on an CytoFLEX6 flow cytometer with CytExpert software as recommended to the manufacturer’s instruction. At the end of study, mice were euthanized. The spleens were excised, photographed, then fixed in 4%paraformaldehyde, paraffin-embedded and stained with hematoxylin and eosin.
- FITC mouse anti-human CD5 BD Pharmingen, USA
- Example 1 LRP1 was highly expressed in leukemia patients
- HCD congenital heart disease
- HCIPs high-confidence candidate interacting proteins
- the canonical Notch signaling pathway relies on the ligand binding to its receptors, making the modulation of ligand activity a critical step for the precise regulation of Notch signaling activation.
- Increasing studies have demonstrated the critical role of ligand endocytosis in Notch activation. Therefore, it is essential to unveil the comprehensive regulation of the ligand-dependent Notch pathway, promoting the development of therapeutic targets in Notch-related diseases.
- the Notch signaling pathway is highly conserved in various species ranging from C. elegans and Drosophila to mammals. We looked whether the regulatory mechanism of LRP1 in the Notch pathway is also conserved in other organisms. First, we knocked down the LRP1 levels in C. elegans ( Figure 3A) and we indeed observed a small portion of animals with abnormal vulva phenotypes when lrp-1 is knocked down by RNAi ( Figure 3B and 3C) . However, there are much larger percentages showing molting defects and the bubble-like phenotype occurring randomly in the whole body of lrp-1 RNAi animals (which theoretically includes the bubble-like phenotypes in or around vulva) ( Figure 3B) .
- Example 5 LRP1 positively regulates Delta and Notch signaling in Drosophila
- LRP1 reduction in the Drosophila eye imaginal disc or in the posterior region of the wing imaginal disc in lrp1 mutant clones or using two independent LRP1 RNAi strains all significantly decreased the level of endogenous Delta (Dl) , the Drosophila orthologue of DLL3 ( Figures 3F-G’ and 4A-C” ) .
- LRP1 knockdown in the posterior region of wing discs reduced endogenous Cut expression, a classic Notch target gene (Figures 3K-M’) .
- Notch signaling has been linked to multiple types of leukemia, including ALL and acute myeloid leukemia (AML) .
- ALL acute myeloid leukemia
- AML acute myeloid leukemia
- LRP1 functions in leukemia pathogenesis by regulating the Notch pathway.
- LRP1 is highly expressed in several leukemia cell lines, including the NOTCH1 wild-type T-ALL cell line HSB2 and the AML cell line K562 ( Figure 4A) , as well as leukemia patients ( Figure 1) .
- LRP1 is an endocytic receptor that internalizes a vast number of ligands, including lipoproteins, and could therefore play a critical role in cellular activity.
- LRP1-deficient cells were normally viable. Comparing with wild-type cells, no differences in viability and apoptosis were recorded, as measured by LDH release assays ( Figure 5A) , Caspase3 activation and Annexin V/PI staining ( Figure 5B-5D) . Also, we examined whether the phenomenon observed in LRP1 KO cells were due to the impaired cholesterol uptake considering its role in lipid metabolism.
- Notch intracellular domain 1 (NICD1) , the active form of NOTCH1, in LRP1-KO HSB2 and K562 cell lines and investigated the tumorigenesis ability of these cell lines both in vitro and in vivo.
- Reconstitution with NICD1 fully rescued colony formation ( Figures 6A-D) and xenograft tumor growth ( Figures 6E-G) in LRP1 KO cells.
- NICD1 Notch intracellular domain 1
- Example 7 LRP1 antagonist RAPm6 inhibits tumorigenesis in human leukemia cells, mouse xenografts and leukemia models
- LRP1 Since LRP1 positively regulates Notch signaling and leukemia tumorigenesis, it may serve as a therapeutic target for Notch signaling-related LRP1-overexpressing cancers.
- Alpha-2-macroglobulin receptor-associated protein (LRPAP1) is a known LRP1 antagonist that has previously been used to block LRP1-related blood-brain barrier opening in a mouse model.
- RAPm6 whose amino acid sequence has been optimized to increase its stability, from Escherichia coli ( Figure 7A) and found that RAPm6 interacts with LRP1 ⁇ ( Figure 7B) .
- RAPm6 treatment significantly decreased Notch target gene expression (Figure 7C) , the cell viability of several leukemia cell lines (Figure 7D) , anchorage-independent colony formation (Figures 7E-H) , and xenograft tumor growth ( Figures 7I-K) without significantly affecting mouse weight ( Figure 7L) .
- RAPm6 treatment did not elicit a prominent effect on xenograft tumor growth of Notch-low MDA-MB-231 cells ( Figures 6M and 6N) , indicating that RAPm6 treatment specifically targets Notch signaling-related cancers.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Urology & Nephrology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
Abstract
Description
Claims (20)
- A method for treating Notch signaling-dependent disease in the subject with a LRP1 inhibitor.
- The method of claim 1, wherein the LRP1 inhibitor is a polypeptide antagonist specifically against LRP1, an RNA polynucleotide specific to LRP1, or a small molecule compound inhibitor specific to LRP1.
- The method of claim 2, wherein polypeptide antagonist is LRPAP1 or LRPAP1 derivative thereof that can bind to LRP1 on the cell surface and prevent ligands from its binding .
- The method of claim 3, wherein the LRPAP1 is a polypeptide comprising:1) an amino acid sequence of SEQ ID NO: 1 or 2;2) an amino acid sequence at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity to SEQ ID NO: 1 or 2; or3) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 1 or 2,the LRPAP1 can bind to LRP1 on the cell surface, preventing ligands from its binding.
- The method of claim 3, wherein the LRPAP1 derivative is a polypeptide comprising:1) an amino acid sequence of SEQ ID NO: 3;2) an amino acid sequence an amino acid sequence with at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity to SEQ ID NO: 3, or3) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 3,the LRPAP1 derivatives can bind to LRP1 on the cell surface, preventing ligands from its binding.
- The method of claim 3, wherein the LRPAP1 derivative is a polypeptide comprising:an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence with at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity to SEQ ID NO: 4, or an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 4.
- The method of claim 6, wherein the LRPAP1 derivative is a polypeptide comprising SEQ ID NO: 4
- The method of any one of claim 4-6, wherein the LRPAP1 or LRPAP1 derivative is a polypeptide with or without a tag.
- The method of claim 8, wherein the tag is selected from c-Myc, His, HA, GST, MBP, Flag, and Arg6.
- The method of any one of claim 4-6, wherein the LRPAP1 or LRPAP1 derivative is a polypeptide modified by PEG.
- The method of claim 2, wherein polypeptide antagonist is an antibody against LRP1.
- The method of claim 2, wherein the RNA polynucleotide is selected from siRNA, shRNA, guide RNA, and miRNA.
- The method of claim 9, wherein the guide RNA is SEQ ID NO: 5 (TGGAGGACAAGATCTACCGC) .
- The method of claim 1, wherein the Notch signaling-dependent disease is selected from leukemia, myeloma, lymphoma, breast cancer, liver cancer, and lung cancer.
- The method of claim 14, wherein the leukemia is T-acute lymphoblastic leukemia or Chronic lymphocytic leukemia.
- The method of claim 1, wherein the subject is non-human mammal or human.
- The method of claim 1, wherein the disease is a metastatic cancer.
- A method of screening medicines for treating Notch signaling-dependent disease using LRP1 as the target, the method comprising: observing the effect of candidate medicine on the expression or activity level of LRP1, if the candidate medicine can inhibit expression or activity level of LRP1, then it indicates that the candidate medicine is a potential medicine for treating Notch signaling-dependent disease.
- The method of claim 18, wherein the Notch signaling-dependent disease is selected from leukemia, myeloma, lymphoma, breast cancer, liver cancer, and lung cancer.
- The method of claim 19, wherein leukemia is T-acute lymphoblastic leukemia or Chronic lymphocytic leukemia.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP22832215.2A EP4362965A1 (en) | 2021-07-02 | 2022-07-01 | Use of a lrp1 inhibitor in treating notch signaling-dependent disease |
CN202280059844.6A CN117940148A (en) | 2021-07-02 | 2022-07-01 | Use of LRP1 inhibitors in the treatment of Notch signaling dependent diseases |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNPCT/CN2021/104314 | 2021-07-02 | ||
CN2021104314 | 2021-07-02 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023274403A1 true WO2023274403A1 (en) | 2023-01-05 |
Family
ID=84690486
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2022/103347 WO2023274403A1 (en) | 2021-07-02 | 2022-07-01 | Use of a lrp1 inhibitor in treating notch signaling-dependent disease |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP4362965A1 (en) |
CN (1) | CN117940148A (en) |
WO (1) | WO2023274403A1 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100055712A1 (en) * | 2008-06-27 | 2010-03-04 | Washington University | Oligopeptides for treatment of osteoporosis and other bone diseases and methods therefor |
WO2011061236A1 (en) * | 2009-11-17 | 2011-05-26 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. | Pharmaceutical compositions comprising an lrp1 receptor agonist for the treatment of cancer and hiv |
-
2022
- 2022-07-01 EP EP22832215.2A patent/EP4362965A1/en active Pending
- 2022-07-01 WO PCT/CN2022/103347 patent/WO2023274403A1/en active Application Filing
- 2022-07-01 CN CN202280059844.6A patent/CN117940148A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100055712A1 (en) * | 2008-06-27 | 2010-03-04 | Washington University | Oligopeptides for treatment of osteoporosis and other bone diseases and methods therefor |
WO2011061236A1 (en) * | 2009-11-17 | 2011-05-26 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. | Pharmaceutical compositions comprising an lrp1 receptor agonist for the treatment of cancer and hiv |
Non-Patent Citations (4)
Title |
---|
BIAN W ET AL.: "Low-density-lipoprotein-receptor-related protein 1 mediates Notch pathway activationBian W", DEV CELL., vol. 56, no. 20, 8 October 2021 (2021-10-08), XP086843900, DOI: 10.1016/j.devcel.2021.09.015 * |
LIU XIAOCONG, WEN JUN-JIE; GUO JIA-DING; NING YUN-SHAN; LI YAN: "Regulation of Notch Signaling Pathways in Tumor Microenvironment", CHINA CANCER, vol. 29, no. 1, 31 January 2020 (2020-01-31), pages 48 - 54, XP093018200, ISSN: 1004-0242, DOI: 10.11735/j.issn.1004-0242.2020.01.A007 * |
MENG HE, ZHANG XIAOJIE, LEE SOO JUNG, STRICKLAND DUDLEY K., LAWRENCE DANIEL A., WANG MICHAEL M.: "Low Density Lipoprotein Receptor-related Protein-1 (LRP1) Regulates Thrombospondin-2 (TSP2) Enhancement of Notch3 Signaling", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 285, no. 30, 14 March 2010 (2010-03-14), US , pages 23047 - 23055, XP093018201, ISSN: 0021-9258, DOI: 10.1074/jbc.M110.144634 * |
WAN-CHING YEN ET AL.: "Targeting Notch signaling with a Notch2/Notch3 antagonist (tarextumab) inhibits tumor growth and decreases tumor-initiating cell frequency", CLIN CANCER RES., vol. 21, no. 9, 1 May 2015 (2015-05-01), XP055222893, DOI: 10.1158/1078-0432.CCR-14-2808 * |
Also Published As
Publication number | Publication date |
---|---|
EP4362965A1 (en) | 2024-05-08 |
CN117940148A (en) | 2024-04-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Huang et al. | M-Phase Phosphoprotein 9 regulates ciliogenesis by modulating CP110-CEP97 complex localization at the mother centriole | |
Hawryluk-Gara et al. | Vertebrate Nup53 interacts with the nuclear lamina and is required for the assembly of a Nup93-containing complex | |
Dix et al. | Lissencephaly-1 promotes the recruitment of dynein and dynactin to transported mRNAs | |
Chao et al. | SUMOylation of the MAGUK protein CASK regulates dendritic spinogenesis | |
Woodley et al. | S‐acylated Golga7b stabilises DHHC 5 at the plasma membrane to regulate cell adhesion | |
Miao et al. | The ER-localized transmembrane protein TMEM39A/SUSR2 regulates autophagy by controlling the trafficking of the PtdIns (4) P phosphatase SAC1 | |
Krishnan et al. | Rab25 regulates integrin expression in polarized colonic epithelial cells | |
Claudius et al. | Unexpected role of the steroid-deficiency protein ecdysoneless in pre-mRNA splicing | |
Diomede et al. | Expression of A2V-mutated Aβ in Caenorhabditis elegans results in oligomer formation and toxicity | |
Bian et al. | Low-density-lipoprotein-receptor-related protein 1 mediates Notch pathway activation | |
Jaspers et al. | The claudin Megatrachea protein complex | |
Petrosyan et al. | Keratin 1 plays a critical role in golgi localization of core 2 N-acetylglucosaminyltransferase M via interaction with its cytoplasmic tail | |
Oguchi et al. | A comprehensive analysis of Rab GTPases reveals a role for Rab34 in serum starvation-induced primary ciliogenesis | |
Xia et al. | CCDC102B functions in centrosome linker assembly and centrosome cohesion | |
US20050043264A1 (en) | Methods of inhibiting neurodegenerative disease | |
JP2015502332A (en) | Method for diagnosis, prognosis prediction or treatment of neurodegenerative diseases | |
Hirst et al. | The role of cargo proteins in GGA recruitment | |
WO2023274403A1 (en) | Use of a lrp1 inhibitor in treating notch signaling-dependent disease | |
Valenti et al. | The increase in maternal expression of axin1 and axin2 contribute to the zebrafish mutant ichabod ventralized phenotype | |
WO2020045349A1 (en) | Agent for activating or suppressing cell-to-cell communication through functional protein, antibody or aptamer, method for screening for said agent, fusion protein, nucleic acid encoding said fusion protein, recombinant vector, transformed host cell, exosome function modulator, and exosome | |
Masilamani et al. | Calpain-mediated cleavage generates a ZBTB18 N-terminal product that regulates HIF1A signaling and glioblastoma metabolism | |
Ryniawec et al. | Cep104 is a component of the centriole distal tip complex that regulates centriole growth and contributes to Drosophila spermiogenesis | |
JP2007505825A (en) | Use of eukaryotic genes that influence cell cycle control or cell cycle progression in the diagnosis and treatment of proliferative diseases | |
US20230374518A1 (en) | Marf/mfn modulators and uses thereof | |
Panda | Role of the Rcd4: Ana3 sub-complex in Drosophila centriole duplication |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22832215 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2022832215 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2022832215 Country of ref document: EP Effective date: 20240202 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 202280059844.6 Country of ref document: CN |